In scanning microscopy, a specimen is illuminated with a light beam in order to observe the detected light, constituting reflected or fluorescent light, emitted by the specimen. The focus of an illuminating light beam is moved in a specimen plane by means of a controllable beam deflection device, generally by tilting two mirrors; the deflection axes are usually perpendicular to one another, so that one mirror deflects in the X direction and the other in the Y direction. Tilting of the mirrors is brought about, for example, by means of galvanometer positioning elements. The power level of the detected light coming from the specimen is measured as a function of the position of the scanning beam. The positioning elements are usually equipped with sensors to ascertain the present mirror position. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen passes through the beam splitter and then arrives at the detectors. In confocal scanning microscopy in particular, a specimen is scanned in three dimensions with the focus of a light beam.
A confocal scanning microscope generally comprises a light source, a focusing optical system with which the light of the source is focused onto an pinhole (called the “excitation pinhole), a beam splitter, a beam deflection device for beam control, a microscope optical system, a detection pinhole, and the detectors for detecting the detected or fluorescent light. The illuminating light is coupled in via a beam splitter. The fluorescent or reflected light coming from the specimen travels by way of the beam deflection device back to the beam splitter, passes through it, and is then focused onto the detection pinhole behind which the detectors are located. This detection arrangement is called a “descan” arrangement. Detected light that does not derive directly from the focus region takes a different light path and does not pass through the detection pinhole, so that a point datum is obtained which results, by sequential scanning of the specimen, in a three-dimensional image. A three-dimensional image is usually achieved by acquiring image data in layers.
The published German Patent Application DE 198 27 140 A1 discloses a laser scanning microscope having an AOTF in the laser coupling-in beam path for line selection and in order to attenuate laser lines.
The published German Patent Application DE 199 06 757 A1 discloses an optical arrangement in the beam path of a light source suitable for fluorescent excitation, preferably in the beam path of a confocal laser scanning microscope, having at least one spectrally selective element for coupling the excitation light of at least one light source into the microscope and for blocking the excitation light scattered and reflected at the specimen, or the excitation wavelength, out of the light coming from the specimen via the detection beam path. For variable configuration with a very simple design, the arrangement is characterized in that excitation light of a differing wavelength can be blocked out by means of the spectrally selective element. Alternatively, an optical arrangement of this kind is characterized in that the spectrally selective element can be set to the excitation wavelength that is to be blocked out. Also stated in the aforementioned document is the fact that the spectrally selective element can be embodied as an acoustooptical tunable filter (AOTF) or an acoustooptical deflector (AOD).
The published German Patent Application DE 198 59 314 A1 discloses an arrangement of a light-diffracting element for the separation of excitation light and emitted light in a microscope beam path, preferably in a confocal microscope, and in particular in a laser scanning microscope, in which context both the excitation light and the emitted light pass through the light-diffracting element and at least one wavelength of the excitationlight is affected by diffraction, while other wavelengths emitted by the specimen pass through the element unaffected and are thereby spatially separated from the excitation light. The arrangement contains an AOTF whose low bandwidth of approx. 2 nm is presented as a particular advantage.
The published German Patent Application DE 198 53 669 A1 discloses an ultrashort pulse source having a controllable multiple-wavelength output, which is utilized in particular in a multi-photon microscope. The system comprises an ultrashort pulsed laser for generating ultrashort optical pulses of a fixed wavelength, and at least one wavelength conversion channel.
U.S. Pat. No. 6,097,870 discloses an arrangement for generating a broadband spectrum in the visible and infrared spectral region. The arrangement is based on a microstructured fiber into which the light of a pump laser is coupled. In the microstructured fiber, the pump light is broadened as a result of nonlinear effects. “Photonic band-gap material” or “photonic crystal fibers,” “holey fibers,” or “microstructured fibers” are also used as the microstructured fiber. Embodiments as a “hollow fiber” are also known.
A further arrangement for generating a broadband spectrum is disclosed in the publication of Birks et al., “Supercontinuum generation in tapered fibers,” Opt. Lett. Vol. 25, p. 1415 (2000). A conventional light-guiding fiber having a fiber core that exhibits a taper at least along a portion is used in the arrangement. Light-guiding fibers of this kind are known as “tapered fibers.”
The PCT application having the publication number WO 00/04613 discloses an optical amplifier whose gain is adjustable as a function of wavelength. Also disclosed in the aforesaid publication is a fiber light source based on this principle.
Arc lamps are known as broadband light sources, and are used in many sectors. One example that may be mentioned here is U.S. Pat. No. 3,720,822 “Xenon photography light,” which discloses a xenon arc lamp for illumination in photography.
In microscopy in particular, spectrally broadband light sources with high light density are important for the illumination of microscopic preparations. These sources can be used flexibly, however, only if illuminating light of the desired wavelength or wavelength spectrum can be easily and flexibly selected out of the emission spectra of the light sources. Arrangements having fixed color filters or dichroic filters do not offer sufficient universality, and are complex and cumbersome in terms of handling. Arrangements having acoustooptical components offer a flexible selection only of a few narrow lines, also offering insufficient flexibility because the lines are arranged serially. The same disadvantages exist in the context of selecting universal wavelengths or wavelength regions of the detected light.